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Diindolylmethane Derivatives – Potent Agonists of the Immunostimulatory Orphan G Protein-Coupled Receptor GPR84 Thanigaimalai Pillaiyar, Meryem Köse, Katharina Sylvester, Heike Weighardt, Dominik Thimm, Gleice Borges, Irmgard Förster, Ivar von Kügelgen, and Christa E Müller J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01593 • Publication Date (Web): 13 Apr 2017 Downloaded from http://pubs.acs.org on April 13, 2017

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Journal of Medicinal Chemistry

Diindolylmethane Derivatives – Potent Agonists of the Immunostimulatory Orphan G Protein-Coupled Receptor GPR84 Thanigaimalai Pillaiyar1≠, Meryem Köse1≠, Katharina Sylvester1, Heike Weighardt2, Dominik Thimm1, Gleice Borges1, Irmgard Förster2, Ivar von Kügelgen3 and Christa E. Müller1* 1

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der

Immenburg 4, D-53121 Bonn, Germany 2

Life and Medical Sciences (LIMES) Institute, Immunology and Environment, University of Bonn,

Carl-Troll-Straße 31, 53115 Bonn, Germany 3Department

≠

of Pharmacology and Toxicology, University of Bonn, 53105 Bonn, Germany

Authors contributed equally

*Corresponding author

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KEYWORDS: Agonist, β-Arrestin, Biased Signalling, Diindolylmethane, Free fatty acid receptor, G protein-coupled receptors, GPR84, GPR40, GPR120, Orphan receptor, Synthesis, Structure-activity relationships

ABSTRACT: The Gi protein-coupled receptor GPR84, which is activated by (hydroxy)fatty acids, is highly expressed on immune cells. Recently 3,3’-diindolylmethane was identified as a heterocyclic, non-lipid-like GPR84 agonist. We synthesized a broad range of diindolylmethane derivatives by condensation of indoles with formaldehyde in water under microwave irradiation. The products were evaluated at the human GPR84 in cAMP and β-arrestin assays. Structure-activity relationships (SARs) were steep. 3,3’-Diindolylmethanes bearing small lipophilic residues at the 5- and/or 7-position of the indole rings displayed the highest activity in cAMP assays, the most potent agonists being di-(5-fluoro1H-indole-3-yl)methane (38, PSB-15160, EC50 80.0 nM) and di-(5,7-difluoro-1H-indole-3-yl)methane (57, PSB-16671, EC50 41.3 nM). In β-arrestin assays SARs were different indicating biased agonism. The new compounds were selective versus related fatty acid receptors and the arylhydrocarbon receptor. Selected compounds were further investigated and found to display an ago-allosteric mechanism of action and increased stability in comparison to the lead structure.


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INTRODUCTION G protein-coupled receptors (GPCRs) constitute the largest known eukaryotic family of integral membrane proteins with more than 800 human genes that encode for GPCRs. It has been estimated that about one third of all modern drugs are targeting members of this receptor family.1 Approximately 50 % are non-olfactory receptors and about 100 of these are poorly characterized orphan receptors whose natural ligand and physiological functions are still unknown or unconfirmed.2 These orphan receptors are, nevertheless, expected to play important (patho)physiological roles. In the last decade an increasing number of GPCRs has been de-orphanized and it was found that free fatty acids (FFAs) act as ligands for several GPCRs, i.e., GPR40, GPR41, GPR43, GPR120 and GPR84.3-8 FFAR1 (GPR40), a receptor for long chain free fatty acids, enhances insulin secretion in pancreatic β-cells, among other functions.3,6 FFAR2 (GPR43) and FFAR3 (GPR41) are stimulated by short chain fatty acids and are involved in energy regulation in adipocytes.4,7 FFAR4 (GPR120) is a receptor for long chain free fatty acids mediating glucagon like peptide-1 secretion.5 GPR84 is an exceptional fatty acid-activated receptor which is predominantly present on immune cells.9,10 In the periphery, GPR84 is mainly expressed in bone marrow, spleen, lung, and peripheral blood leukocytes. In the CNS, GPR84 expression is restricted to microglia. In addition, GPR84 is expressed in adipose tissues.10,11 Table 1 provides a summary of the fatty acid receptors, their coupling and important functions.3,4,6,12-14



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Table 1. G protein-coupled fatty acid receptors, their ligands, coupling and physiological functions Receptor

G protein

Important physiological function

coupling

FFAR1

Gq/11

agonists

(fatty acids)

Glucose-dependent insulin release, inhibition of Long chain, C12osteoclastogenesis15,16 and regulation of

(GPR40)

Potent

C18

taste preference for fatty acids17 FFAR2

Gq/11, Gi/o

(GPR43) FFAR3

Gi/o

(GPR41) FFAR4

Gq/11

Immune function, hematopoiesis, mast cell

Short chain,

activity, and adipogenesis.18

C3=C4=C2

Pancreatic peptide YY (PYY) secretion and

Short chain,

glucacon-like peptide 1 (GLP-1) secretion19

C3=C4>>C2

GLP-1 secretion5

Long chain, C12-

(GPR120) GPR84

C16 Gi/o

Immunostimulation,9 proinflammatory,24

Medium chain,

defense against pathogens10

C9-C14

GPR84 was discovered by sequence tag data mining and cloned from a cDNA library prepared from human peripheral blood neutrophils.20,21 The human and mouse GPR84 genes encode for a protein of 396 amino acids in length with 85 % identity.20 GPR84 couples primarily to a pertussis toxin (PTX)sensitive Gi/o pathway in response to FFAs, including hydroxylated FFAs, of medium carbon chain length (C9-C14, MCFA).9 MCFAs, such as decanoic acid (capric acid), undecanoic acid, and dodecanoic acid (lauric acid) were shown to act through GPR84 in RAW264.7 cells, a murine macrophage-like cell line, amplifying the stimulation of lipopolysaccharide (LPS)-induced interleukin12B (IL12B) production.9,22 The pro-inflammatory cytokine IL-12 plays a pivotal role in promoting cellmediated immunity to eradicate pathogens by inducing and maintaining T helper 1 (Th1) responses and inhibiting T helper 2 (Th2) response, which plays an important role in antibody-mediated immunity. The expression of GPR84 in the peripheral immune system and in microglia suggests a role in the regulation of (neuro)inflammatory processes.10 Recent evidence showed that GPR84 deficiency decreased the proliferation of leukemia cells and has therefore been proposed for the treatment of acute ACS Paragon Plus Environment

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myeloid leukemia.23 The expression of GPR84 in adipose tissues may indicate that the receptor plays a role in obesity.24 GPR84 may be a promising drug target for a variety of inflammatory diseases including Alzheimer’s disease,25 neuropathic pain,26 reflux esophagitis,27 and inflammatory bowel disease.28 MCFAs are only weakly potent activators of GPR84. Moreover, these putative endogenous ligands of GPR84, including saturated fatty acids ranging in chain length from C9 to C14, also activate the free fatty acid (FFA) receptors FFAR1 (GPR40) and FFAR4 (GPR120).6,29,30 Suzuki et al. reported that 2hydroxy- and 3-hydroxy-fatty acids were more potent GPR84 agonists than non-hydroxylated fatty acids. In the same study, 6-n-octylaminouracil (6-OAU, Figure 1), which displays a lipid-mimetic structure, was reported as a synthetic agonist of human GPR84 with an EC50 value of 512 nM determined in [35S]GTPɣS binding assays.13,29,31 More recently, the structurally related 2(hexylthio)pyrimidine-4,6-diol (Figure 1) and its derivatives32,33 were identified as GPR84 agonists. In addition, the natural product embelin (embelic acid, 3-undecyl-2,5-dihydroxy-1,4-benzoquinone, Figure 1) has also been reported to be a GPR84 agonist with moderate potency.34 Embelin is a secondary metabolite originally isolated from the plant Embelia ribes (Primulaceae), which was reported to have anthelmintic, antifertility, antitumor, anti-apoptotic, antimicrobial, analgesic, and anti-inflammatory activity.35-37 Besides GPR84 activation, embelin has several other activities including inhibition of Xlinked inhibitor of apoptosis protein (XIAP, IC50 4.1 µM),38 activation of caspase 9,38 and anti-oxidant properties.39 Another natural product-derived GPR84 agonist, 3,3’-diindolylmethane (1, Figure 1) was discovered,40 which possibly acts via an allosteric binding site since its structure does not resemble fatty acids and is not lipid-like.41 1 is the major metabolite of indole-3-carbinol which is a break-down product of glucobrassicin, a glucosinolate that is present in high concentrations in some vegetables such as broccoli.42 The compound was previously reported to activate the arylhydrocarbon receptor (AhR), a ligand-activated transcription factor associated with gastric carcinogenesis,43 as well as estrogen



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receptors44 at concentrations of around 10 µM and higher. 1 Showed anti-obesity effects in mice.45 and displayed anti-cancer activity in vitro and in vivo.46

Figure 1. Selected GPR84 agonists

While most GPR84 agonists contain long alkyl chains and are thus highly lipophilic, 1 is a small heterocyclic molecule consisting of indole rings, which are found in many perorally applied drug molecules.47-49 In the present study, we therefore selected 1 as a lead molecule and studied its structureactivity relationships (SARs) with the goal to improve its potency. The synthesized compounds were evaluated at the human GPR84 in cAMP accumulation as well as β-arrestin recruitment assays.


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RESULTS AND DISCUSSION Compound design In order to explore the SARs of diindolylmethane derivatives at GPR84, the following modifications were targeted (Figure 2): (A) substitution of the indole rings in various positions including symmetrical and unsymmetrical substitution patterns, (B) mono-substitution of the 3,3’-methylene linker (C10) by alkyl and aryl residues, (C) replacement of the 3,3’-methylene linker (CH2) by a carbonyl group, (D) oxidation of the diindolylmethane ring system and (E) subsequent partial reduction yielding compounds E.

Figure 2. Modifications of 3,3’-diindolylmethane (1)

Chemistry It is well known that indoles react with aliphatic or aromatic aldehydes and ketones to produce azafulvenium salts; these can undergo further addition with a second indole molecule to afford diindolylmethanes. A number of synthetic methods for the preparation of 3,3’-diindolylmethanes have been reported in the literature using protic acid (e.g. HCl),50-52 Lewis acids (e.g. AlCl3, BF3, I2),53,54 or different lanthanide triflates or chlorides.55-58 Recently, benzoic acid59 in water, sodium dodecylsulfate (SDS)60 as a surfactant in water, oxalic acid and N-acetyl-N,N,N-trimethylammonium bromide (CTAB) ACS Paragon Plus Environment


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in water,61 metal triflate in ionic liquid,57 in ionic liquids with or without Fe(III) salts62,63 were reported to provide efficient conditions for the transformation of indoles to diindolylmethanes. However, there are still a number of drawbacks in the available catalytic systems including the requirement of large55,64or stoichiometric amounts of catalysts,65 long reaction times55,59,64 and low yields.66 Organic reactions in water as a reaction medium offer advantages, including low cost, safe handling and environment-compatibility. There are few reports on the synthesis of diindolylmethanes in water via the condensation of indole with aliphatic or aromatic aldehydes.59,60 This led us to attempt the reaction of indole with various aldehydes in aqueous media under microwave irradiation. In our initial attempt, the reaction of indole (2, 10 mmol) with formaldehyde (37 %, 5 mmol) in water at 100 oC for 5 minutes was selected as a model reaction (entry 1, Table 2). The desired product 1 was isolated in reasonable quantity (43 %) without a catalyst. In order to improve the yield, reaction time, temperature and catalyst were optimized (Table 2). As shown in entries 2–4, the yield was gradually improved by increasing the reaction time, and the best result was obtained for reactions at 100 oC for 20 min (entry 4, 98 % yield). The reaction temperature is one of the most important criteria as the reduction of the temperature from 100 oC to 75 or 50 oC in a 30 min reaction resulted in low yield (entry 5 and 6). The reaction was also studied in the presence of acids as catalysts, concd. H2SO4 or CH3COOH, at 100 oC for 30 min; however, these conditions resulted in poor yields (20 % for entry 7 and 30 % for entry 8, Table 2).


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Table 2. Microwave-assisted synthesis of 3,3’-diindolylmethane in water under different conditions

Entry

Catalyst

Temp (oC)

Time (min)

Power (W)

Yield (%)

1

-

100

5

105

43

2

-

100

10

105

84

3

-

100

15

105

91

4

-

100

20

105

98

5

-

75

30

75

42

6

-

50

30

75

28

7

concd. H2SO4 100

20-30

105

20

8

CH3CO2H

20-30

105

30

100

The optimized conditions were subsequently used for the synthesis of a series of diindolylmethane derivatives (Scheme 1). The desired products 32-60 were obtained from the starting indoles 2-31 in very good to excellent yields (84-98 % isolated). Detailed reaction conditions and purities of the products (32-60) are summarized in Table 3. It should be noted that the microwave-assisted reactions were completed within only 20-30 min for most of the derivatives.

Scheme 1. Synthesis of 3,3’-diindolylmethane derivatives 32-60a

a

Reagents and conditions: (a) H2O, 100 oC, microwave, 20–180 min, yield 45–98 %. b For R1, R2 and R3

see Table 3. ACS Paragon Plus Environment


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Table 3. Reaction times, yields, melting points and purities of 3,3’-diindolylmethane derivatives

Compd.

R1

R2

R3

Time (min)

Yield (%)

160

H

H

H

20

98

3267

4-CH3

H

H

25

93

3368

4-OCH3

H

H

25

87

34

4-F

H

H

25

82

35

4-Cl

H

H

25

86

3667

5-CH3

H

H

25

91

3767

5-OCH3

H

H

25

89

3867

5-F

H

H

30

90

3967

5-Cl

H

H

20

92

4067

5-Br

H

H

25

87

4169

5-CN

H

H

29

92

4269

5-NO2

H

H

35

90

43

5-COOMe

H

H

25

84


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44

5-CHO

H

H

25

85

45

5-COOH

H

H

25

79

46

5-OCH2Ph

H

H

35

75

4768

6-CH3

H

H

25

88

4868

6-OCH3

H

H

25

82

4968

6-F

H

H

30

87

5068

6-Cl

H

H

25

90

51

7-OCH3

H

H

25

93

52

7-F

H

H

25

85

53

4-Cl, 6-Cl

H

H

30

83

54

5-F, 6-Cl

H

H

30

76

55

4-F, 5-F

H

H

20

87

56

5-F, 6-F

H

H

20

83

57

5-F, 7-F

H

H

30

80

5870

H

H

CH3

25

89

5968

CH3

CH3

H

25

82

25

82

6068

see structure above

Surfactants can play an important role as emulsifiers in water-mediated organic reactions.59,60,71 In order to avoid solubility problems, we used sodium dodecyl sulfate (SDS) in our next optimization step ACS Paragon Plus Environment


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reacting indoles with various aliphatic and aromatic aldehydes under microwave irradiation in water. Indole 2 was treated with the appropriate aldehyde in the presence of 10 % SDS in water (5 mL), and the resulting white turbid mixture was irradiated in a microwave oven at 100 oC for 30-40 min (Scheme 2A). As shown in Table 4, both, aliphatic (61-66) and aromatic aldehydes (67-74), afforded good yields of the corresponding products (75-88).60,71-76 Diindolylmethanone 92 was synthesized using a published procedure as indicated in Scheme 2B.77 Indole-3-carboxylic acid (89) was converted to the corresponding acid chloride 90 by reaction with thionyl chloride in dichloromethane at 45 oC for 1 h. The resulting acid chloride was subsequently treated with indole (2) in the presence of zirconium(IV) chloride in dichloroethane at room temperature for 4 h to produce the desired product 91.


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Scheme 2. Synthesis of diindolylmethane derivatives 75-88a and 91b A)

B)

a

Reagents and conditions: (a) sodium dodecyl sulfate (SDS), H2O, 95–100 oC, microwave (MW), 30-40

min, yield 72–95 %. For R see Table 5; b(a) SOCl2, dichloroethane (DCE), 40 oC, 1 h; b) ZrCl4, DCE, 0 o

C to rt, 4 h, 69 %.

Table 4. Reaction times, yields, melting points and purities of products 75–88 and 91

R1

Reaction time (min)

Yield (%) (lit. yield)

7572

H

30

89

76

4-F

30

83

Compd.

R



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77

H

30

80

7873

H

30

84 (73)

7974

H

30

8075

H

35

72 (21)

8152

H

40

85 (65)

8252

H

40

95 (80)

83

H

40

93

8452

H

40

87 (55)

85

H

40

80

86

H

40

91

8776

H

40

94 (89)

8852

H

40

90 (60)


89 (62)

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9177

See structure above

40

69

Unsymmetrical 3,3’-diindolylmethanes were synthesized through an intermolecular Pummerer reaction using indole as a nucleophile.78 As shown in Scheme 3, indole was treated with dimethyl sulfoxide (DMSO) and trifluoroacetic acid anhydride in 1,4-dioxane at 0 oC, and the mixture was heated at 80 oC for 30 min to obtain intermediate 92, which was subsequently converted to the unsymmetrical 3,3’diindolylmethanes 93–97 by heating it with substituted indole (8, 9, 14 or 29) and copper acetate for 2 h. Product 97 was obtained in an excellent yield of 99 % after hydrolysis of the dimethyl ester 96, which was achieved by treatment with 2-N sodium hydroxide in ethanol under reflux for 2 h. Yields, melting points and purities of synthesized derivatives are collected in Table 5.



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Scheme 3. Synthesis of unsymmetrical 3,3’-diindolylmethanes 93-98a

a

Reagents and conditions: (a) DMSO, trifluoroacetic anhydride (TFAA), dioxane, 0 oC then 80 oC, 30

min; (b) Cu(OAc)2, dioxane, 80 oC, 1 h, yield 35–47%; c) 2 N NaOH, ethanol, 100 oC, 1 h, yield 99%. For R1, R2 see Table 5.

Table 5. Yields, melting points and purities of synthesized unsymmetrically substituted 3,3’diindolylmethanes (93–97)

Compd.

R1

R2

Yield (%)

93

H

CH3

40

94

OCH3

H

47

95

F

H

41

96

CO2CH3

H

35

97

CO2H

H

99


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2-Oxoindole derivatives were synthesized as illustrated in Scheme 4. 2-Oxoindole derivative 98 or 99 was reacted with the appropriate aldehyde (100-103) in absolute ethanol in the presence of piperidine at 65 oC for 2 h to produce α,β-unsaturated indole-2-one derivatives 104-109.79-83 These products were reduced in the presence of sodium borohydride at 65 oC for 2 h to yield the corresponding saturated indole-2-one derivatives 110-113.79

The structures of the synthesized compounds were confirmed by 1H,

13

C, and/or

13

C attached protein

test (13Capt) NMR spectroscopy, a common experiment to assign C-H multiplicities in 13C NMR spectra. In addition, HPLC analysis coupled to electrospray ionization mass spectrometry (LC/ESI-MS) was performed, which was also used to determine the purity of the compounds.

Scheme 4: Synthesis of unsaturated and saturated 2-oxoindole derivatives 104-109 and 110–113a

a

Reagents and condition: (a) piperidine, ethanol, 65 oC, 2 h, yield 69–80%; (b) NaBH4, ethanol, 65 oC, 2

h, yield 73–95%. For R1, R2 see Table 6.



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Table 6. Yields, melting points and purities of synthesized 2-oxoindole derivatives 104-113

Compd.

R1

R2

Yield (%)

10480

H

H

76

105

H

F

69

10673

H

OCH3

73

10781

F

F

85

108

F

OCH3

80

10983

See structure above

73

11082

H

OCH3

89

111

F

F

96

112

F

OCH3

91

113

See structure above


95

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Pharmacological evaluation All compounds were initially investigated in cAMP accumulation assays at a concentration of 10 µM for their potency to inhibit forskolin (10 µM) induced cAMP accumulation in Chinese hamster ovary (CHO) cells stably expressing the Gi protein-coupled human GPR84. For compounds leading to more than 50 % inhibition of cAMP accumulation, full concentration-response curves were determined and EC50 values were calculated (see Table 7 and 8). In order to determine the compounds’ efficacy, their maximal effects were compared to the maximal signal induced by decanoic acid (100 µM; EC50 7.42 µM). Compounds that did not show agonistic activity were tested for their potency to antagonize receptor activation by decanoic acid (20 µM) at a concentration of 10 µM. To exclude GPR84independent effects, the most potent compounds were additionally investigated in the parent nonGPR84-transfected CHO cell line. Selected compounds were further investigated in GPR84-dependent β-arrestin recruitment assays using the β-galactosidase fragment complementation technology (Pathhunter, DiscoverX)84,85 (see Table 7 and 8). We additionally confirmed the allosteric interaction of diindolylmethane and its optimized derivative 57 with GPR84 in cAMP accumulation assays applying different combinations of the orthosteric agonist decanoic acid and diindolylmethanes. To investigate GPR84 selectivity, potent agonists were evaluated at those human free fatty acid receptor subtypes that show overlapping agonist specificity with GPR84 and are also activated by dodecanoic acid, namely FFAR1 and FFAR4 (see Table S2 and S3). The Gq protein-coupled human FFAR1 was retrovirally expressed in 1321N1 astrocytoma cells and calcium mobilization assays were performed. The human FFAR4 was expressed in a CHO cell line suitable to perform β-arrestin recruitment assays using the βgalactosidase complementation assay technology. All compounds were tested for agonistic as well as antagonistic activity at FFAR1 and FFAR4 in a final concentration of 10 µM. Selected diindolylmethane derivatives were further evaluated for activation of the arylhydrocarbon receptor, the most prominent target of lead compound 1.



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Table 7. Potency of symmetrical 3,3’-diindolylmethane derivatives 32–60 as agonists at the human GPR84 determined in cAMP accumulation and β-arrestin recruitment assays

human GPR84 Compd.

1

R

2

R

3

R

cAMP assaya

β-arrestin assay

EC50 ± SEM (µM) (or percent receptor activation at 10 µM) [efficacy]b

EC50 ± SEM (µM) (or percent receptor activation at 10 µM) [efficacy]c

Decanoic acid

7.42 ± 0.40 [100 %]*

6.08 ± 0.36 [92 %]#

Embelin

0.795 ± 0.168 [86 %]§

0.400 ± 0.142 [100 %]$

1

H

H

H

0.252 ± 0.088 [125 %]

1.64 ± 0.81 [60 %]

32

4-CH3

H

H

0.987 ± 0.235 [112 %]

> 10 (24 ± 8 %)

33

4-OCH3

H

H

≥ 10 (47 ± 17 %)

n.d.*

H

H

0.328 ± 0.005 [110 %]

1.23 ± 0.03 [108 %]

34 (PSB- 4-F 16357) 35

4-Cl

H

H

1.14 ± 0.06 [111 %]

> 10 (25 ± 14 %)

36

5-CH3

H

H

≥ 10 (45 ± 9 %)

≥ 10 (49 ± 6 %)

H

H

0.369 ± 0.025 [119 %]

1.20 ± 0.80 [103 %]

H

H

0.0800 ± 0.0212 [117 %]

4.33 ± 2.12 [125 %]

37 (PSB- 5-OCH3 16105) 38 (PSB- 5-F 15160) 39

5-Cl

H

H

4.96 ± 3.08 [86 %]

n.d.

40

5-Br

H

H

3.44 ± 0.29 [106 %]

n.d.

41

5-CN

H

H

≥ 10 (49 %)

n.d.

42

5-NO2

H

H

> 10 (31 %)

n.d.

43

5-COOMe

H

H

> 10 (11 %)

n.d.

44

5-CHO

H

H

> 10 (-4 %)

n.d.


Page 21 of 70

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45

5-COOH

H

H

≥ 10 (28 %)

>> 10 (-5 ± 6 %)

46

5-OCH2Ph

H

H

> 10 (36 %)

n.d.

47

6-CH3

H

H

> 10 (-22 %)

n.d.

48

6-OCH3

H

H

> 10 (31 %)

n.d.

H

H

0.625 ± 0.070 [89 %]

7.53 ± 2.30 [48 %]

49 (PSB- 6-F 16358) 50

6-Cl

H

H

> 10 (4 %)

5.34 ± 1.84 [118 %]

51

7-OCH3

H

H

> 10 (18 %)

n.d.

H

H

0.113 ± 0.047 [129 %]

6.07 ± 3.37 [99 %]

52 (PSB- 7-F 16381) 53

4-Cl,6-Cl

H

H

≥ 10 (51 %)

n.d.

54

5-F,6-Cl

H

H

10.8 ± 0.50 [83 %]

3.98 ± 0.86 [181 %]

55

4-F,5-F

H

H

0.377 ± 0.030 [123 %]

>> 10 (-2 ± 9 %)

56 (PSB- 5-F,6-F

H

H

0.187 ± 0.053 [121 %]

10.3 ± 5.6 [92 %]

H

H

0.0413 ± 0.0098 [131 %]

5.47 ± 0.13 [385 %]

16586) 57 (PSB- 5-F,7-F 16671) 58

H

H

CH3

> 10 (-37 %)

>> 10 (-3 %)

59

H

CH3

H

≥ 10 (42 %)

> 10 (23 %)

60

see structure above

-

> 10 (-12 %)

2.27 ± 0.42 [115 %]

*n.d., not determined a Inhibition of forskolin (10 µM) induced decrease in cAMP accumulation b Efficacy (Emax) relative to the max. effect of decanoic acid (100 µM) (= 100 %) c Efficacy (Emax) relative to the max. effect of embelin (10 µM) (= 100 %) * Literature value: EC50 = 4.5 ± 0.3 µM9 # Literature value: EC50 = 10 µM84 § Literature value: EC50 = 421 nM85 $ Literature value: EC50 = 12.3 µM84

Structure-activity relationships cAMP accumulation assays The lead compound 1 induced an inhibition of forskolin-induced cAMP accumulation in CHO cells transfected with the human Gi-coupled GPR84 displaying an EC50 252 nM. (see Fig. 4). It was 29-fold ACS Paragon Plus Environment


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Page 22 of 70

more potent than decanoic acid (p=0.0021,**) and showed a higher efficacy (125 %) compared to decanoic acid (set at 100 %) that – in contrast to 1 - did not completely inhibit cAMP formation at the highest concentration used (Fig. 4). We confirmed that 1 had no effect on forskolin-induced cAMP levels in non-transfected CHO cells (see Fig. 4). This clearly proves that the observed effect on cAMP levels was due to GPR84 activation.

When exploring the SARs of diindolylmethane derivatives we initially focused on substitution of the indole rings (Table 7). SAR analysis revealed that many symmetrically substituted indole derivatives were potent GPR84 agonists. However, substitution at several positions of the indole rings resulted in loss of activity, and substitution was only permitted in few positions. These results indicated very steep SARs, clearly not driven by an increase in lipophilicity. The following substituents were introduced at the 4,4’-positions of the indole rings: 4,4’-dimethyl (32), 4,4’-dimethoxy (33), 4,4’-difluoro (34) and 4,4’-dichloro (35). A deleterious effect on activity was observed with these substituents, except for compound 34 (EC50 328 nM) featuring 4,4’-difluoro substitution, which exhibited comparable activity to the lead compound (1). Next, substituents were introduced at the 5,5’-positions yielding compounds 36–46. Among them, the 5,5’-difluoro derivative (38: EC50 80.0 nM) showed high activity being 3-fold more potent than the lead compound 1. 5,5’Dimethoxy substitution (37: EC50 369 nM) conferred moderate potency, whereas 5,5’-dichloro (39: EC50 4960 nM) and 5,5’-dibromo derivatives (40: EC50 3440 nM) displayed only weak potency. Substitution at the 6,6’-positions (compounds 47-50) did not improve agonistic potency, and the small 6,6’-difluoro substituents were best tolerated (compound 49, EC50 625 nM). Based on these results we only prepared two 7,7’-substituted diindolylmethanes with 7,7’-dimethoxy (51) and 7,7’-difluoro (52) derivatives. Interestingly, compound 52 (EC50 113 nM) was found to be 6-fold more potent than the corresponding 6,6’-difluoro-substituted isomer 49 (EC50 625 nM) (p=0.0057, **), and appeared to be slightly more potent than lead compound 1.


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Encouraged by these results our next effort was to introduce multiple halogen substituents with the goal to identify the best combination (compounds 53-57). Chloro substitution at the 4- and 6- positions (53) was not tolerated probably because of the size of the halogen atoms. When we introduced the smaller fluorine atoms, the potency of the resulting compounds (54-57) was much higher. A combination of fluorine atoms in the 5- and 7-positions (57) was found to be the best combination, and in fact compound 57 (EC50 41.3 nM) was the most potent GPR84 agonist of the present series.

The replacement of the indole NH hydrogen atoms of lead structure 1 by methylation yielding the 1,1’dimethyl derivative 58 (EC50 >10 µM) or the 1-methyl derivative 59 (EC50 ≥10 µM, see Table 8) abolished potency of the compound. This confirms that the indole NH functions are very important for interaction with GPR84, presumably by the formation of hydrogen bonds. Substitution of the 2positions as in the 2,2’-dimethyl derivative 59, or introduction of a nitrogen atom as in di-(7azaindolyl)methane (60) was not tolerated. These results obtained from substitution and modification of the indole rings clearly indicated that substituents other than fluoro were not well tolerated and in all cases led to a decrease in agonistic potency at GPR84. The rank order of potency for substituents at various positions of the indole rings is as follows: 5,5’-difluoro (38: EC50 80.0 nM), 7,7’-difluoro (52: EC50 113 nM) > 4,4’-difluoro (34: EC50 328 nM, p=0.0104, *) ≥ 5,5’-dimethoxy (37: EC50 369 nM) > 6,6’-difluoro (49: EC50 625 nM, p=0.0262 *). Only fluoro-substitution in the 7- and particularly in the 5-position improved the potency of the unsubstituted lead structure 1.

In the next set of experiment, we investigated the effects of mono-substitution at the 5-position of only one of the indole rings (Table 8). The following rank order of potency was observed: 5-fluoro (95: EC50 264 nM) > 5-methoxy (94: EC50 1890 nM, p=0.0013, *) > 5-carboxymethyl ester (96: EC50 ≥10 µM) > 5-carboxylic acid (97: EC50 >>10 µM). This means that also in the mono-substituted series only small residues were tolerated in the 5-position, and a fluorine atom (compound 95) was best showing similar potency as the unsubstituted lead structure 1. The potencies of the mono-substituted derivatives 94 (5ACS Paragon Plus Environment


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Page 24 of 70

fluoro) and 95 (5-methoxy) were reduced when compared to the corresponding di-substituted derivatives 37 (p=0.0014, **) and 38 (p=0.0776, ns). These results revealed that symmetrical substitution of 1 is superior to mono-substitution yielding unsymmetrical diindolylmethane derivatives (compare 37/94 and 38/95).

We subsequently examined the effects of substitution at the 3,3'-methylene bridge (C-10) of 1 (Table 8). A large variety of substituents including methyl (75-77), n-propyl (78), n-butyl (79), isopropyl (80), 4tolyl (81), 4-anisyl (82), 4-hydroxyphenyl (83), 4-chlorophenyl (84), 4-fluorophenyl (85), 4-(1,3benzodioxolyl) (86), 3-indolyl (87) and 4-nitrophenyl (88) were introduced. However, none of the substituents was tolerated at that position, and all compounds were inactive. On the other hand, 3,3’diindolylmethanone (91: EC50 553 nM), an oxidized derivative of 1, was found to be almost as potent as the lead structure 1 (EC50 252 nM).


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Table 8. Potency of 3,3’-diindolylmethane derivatives 75-113 as agonists at the human GPR84 determined in cAMP accumulation and β-arrestin recruitment assays

Human GPR84

Compound

R

1

2

R

cAMP assaya

β-arrestin assay

EC50 ± SEM (µM) (or percent receptor activation at 10 µM) [efficacy]b

EC50 ± SEM (µM) (or percent receptor activation at 10 µM) [efficacy]c

Structure A: Substitution of the methylene linker (C10) 75

–

> 10 (-5 %)

> 10 (3 %)

76

4-F

> 10 (5 %)

> 10 (34 %)

77

–

> 10 (-24 %)

2.97 ± 1.03 [67 %]

78

–

> 10 (-24 %)

6.07 ± 0.77 [199 %]

79

–

> 10 (-4 %)

> 10 (-3 %)

80

–

> 10 (16 %)

7.29 ± 0.19 [167 %]

81

–

> 10 (2 %)

> 10 (10 %)

82

–

> 10 (-3 %)

n.d.

83

–

> 10 (7 %)

> 10 (-5 %)

84

–

> 10 (-4 %)

n.d.

85

–

> 10 (8 %)

n.d.



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Page 26 of 70

86

–

> 10 (0 %)

> 10 (7 %)

87

–

> 10 (-10 %)

> 10 (10 %)

88

–

> 10 (6 %)

n.d.

Diindolylmethanone and unsymmetrically substituted diindolylmethane derivatives

Structure B: 91 (PSB16359)

–

0.553 ± 0.141 [104 %]

19.0 ± 8.1 [161 %]

93

–

≥ 10 (43 %)

> 10 (8 %)

94

–

1.89 ± 0.19 [97 %]

6.47 ± 3.20 [110 %]

95 (PSB16244)

–

0.264 ± 0.075 [115 %]

1.08 ± 0.58 [72 %]

96

–

ca. 10 (57 %)

n.d.

97

–

> 10 (5 %)

n.d.

Structure C: Oxidized products of diindolylmethane derivatives (methene bridged) 104

H

> 10 (-17 %)


> 10 (-22 %)

Page 27 of 70

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Journal of Medicinal Chemistry F

105

H

> 10 (-1 %)

n.d.

> 10 (-10 %)

n.d.

> 10 (5 %)

n.d.

N H

106

H F

107

5-F N H

108

5-F

> 10 (7 %)

> 10 (-31 %)

109

H

> 10 (-16 %)

> 10 (-7 %)

Structure D: 2-Oxodihydroindolyl-indolylmethane derivatives

110

H

> 10 (-21 %)

n.d.

> 10 (16 %)

n.d.

F

111

5-F N H

112

5-F

> 10 (-10 %)

n.d.

113

H

> 10 (7 %)

n.d.

a

Inhibition of forskolin (10 µM) induced decrease in cAMP accumulation Efficacy (Emax) related to the max. effect of decanoic acid (100 µM) (= 100 %) c Efficacy (Emax) related to the max. effect of embelin (10 µM) (= 100 %) b

To obtain deeper insight into the SARs of diindolylmethane derivatives, we altered the hydrogen bonding characteristics of 1 by preparing 2-oxyindoles. To this end we prepared a series of unsaturated (104–109) and saturated 2-oxyindole derivatives (110–113). However none of these compounds was



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Page 28 of 70

found to interact with GPR84 (see Table 8) and this might be due to disruption of the planarity of the molecules.

Figure 3 summarizes the structure-activity relationships of the investigated compounds as agonists of the human GPR84: (i) symmetrical diindolylmethane derivatives show the highest potency, (ii) small substituents like methoxy and fluoro are tolerated at C4, C5, and C7 of the benzene rings, e.g., 4,4’difluoro- (34), 5,5’-dimethoxy- (37) and 5-fluoro-diindolylmethane (95) show similar potency as the unsubstituted lead structure, while 5,5’-difluoro- (38), 7,7’-difluoro- (52), and 5,5’,7,7’-tetrafluorodiindolylmethane (57) display enhanced agonistic potency; (iii) substitutions or modifications of the methylene linker are not tolerated.

Figure 3: Structure-activity relationships of diindolylmethane derivatives as agonists at human GPR84

Concentration-response curves for the best two GPR84 agonists, 38 and 57, as well as for the known agonists decanoic acid and diindolylmethane (1) are displayed in Figure 4. The diindolylmethanes were additionally tested at the parent CHO cell line that does not express GPR84. While 1, 38 and 57 show a concentration-dependent inhibition of forskolin-induced cAMP accumulation in CHO cells transfected


Page 29 of 70

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with the human GPR84, no inhibition is observed in non-transfected cells. This proves that the effects observed for the diindolylmethane derivatives are due to GPR84 activation.

Figure 4. Concentration-response curves of selected GPR84 agonists determined in cAMP accumulation assays. Non-transfected CHO cells or CHO cells stably expressing the human GPR84 (CHO-hGPR84) were preincubated with the respective test compounds at the indicated concentrations for 5 min. Then 10 µM forskolin was added and the cells were incubated for additional 15 min. The maximal forskolin-induced cAMP accumulation in the absence of agonist stimulation was defined as 100 %. EC50 values for the investigated agonists were as follows: 38: 80.0 nM, 57: 41.3 nM; decanoic acid: 7420 nM, diindolylmethane: 252 nM. Mean values ± SEM from 3 independent experiments performed in duplicates are shown.

In cAMP assays diindolylmethane (1) showed somewhat higher efficacy (125 %) than the standard agonist decanoic acid (set as 100 %, p=0.0006,

***

). Most potent diindolylmethane derivatives and

analogs displayed efficacies in the same range as 1, e.g. for the most active compounds 38, 52, and 57 it ranged from 117-131 % of the maximal effect observed for decanoic acid. Only the 5,5’-dichloroACS Paragon Plus Environment


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diindolylmethane (39) showed a significantly lower efficacy of 86 % (p=0.0002,

Page 30 of 70 ***

) . These results

indicate that ligands binding to the diindolylmethane binding site induce effects on the receptor conformation which lead to efficient Gi protein coupling.

Compounds which had not exhibited any agonistic activity in cAMP assays were tested for their ability to block GPR84 activation. However, none of the compounds was found to act as an antagonist at GPR84 at a concentration of 10 µM (data not shown).

β-Arrestin assays Selected GPR84 agonists identified in cAMP assays were further evaluated in β-arrestin assays (see Table 8 and 9). We used the β-galactosidase complementation assay technology (DiscoverX®), since it is highly specific for the expressed receptor. The human GPR84 containing a fragment of βgalactosidase, and β-arrestin containing the complementary fragment of the functional enzyme are coexpressed in CHO cells. When GPR84 is activated, β-arrestin is recruited resulting in the formation of a functional enzyme.84,85 This assay hardly produces any artifacts. Moreover we could exclude potential false-positive results by testing the compounds versus another GPCR, the fatty acid receptor FFAR4, expressed in the same system. As a control, we used the agonist embelin which is more suitable as a standard agonist in the β-arrestin assay than decanoic acid since it is more potent in that assay and has a higher efficacy. Both lipidic agonists displayed virtually identical potency in cAMP accumulation and β-arrestin translocation assays, whereas 1 was 7-fold more potent in the Gi-dependent pathway as compared to β-arrestin recruitment, even though the difference was not statiscically significant (p=0.1637, ns). The SARs of diindolylmethane derivatives determined in the β-arrestin assay were quite different from those observed in the cAMP assays. Surprisingly, the two most potent compounds in cAMP assays, 38 and ACS Paragon Plus Environment

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57, activated GPR84 in the β-arrestin assay with only moderate potency, displaying 54-fold (38) and 132-fold (57, p98 %. Mp: 231–233 °C.



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Page 44 of 70

Di-(5-formyl-1H-indole-3-yl)methane (44). The compound was synthesized using indole-5carboxaldehyde (15) and was isolated as a brown solid (85 % yield). 1H NMR (500 MHz, DMSO-d6) δ 11.34 – 11.30 (br s, 2H, NH), 9.93 (s, 2H), 8.18 (d, J = 1.4 Hz, 2H), 7.60 (dd, J = 8.5, 1.5 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 2.2 Hz, 2H), 4.28 (s, 2H). 13Capt NMR (126 MHz, DMSO-d6) δ 192.51, 140.00, 128.40, 127.06, 125.25, 124.23, 121.12, 116.18, 112.22, 20.68. LC-MS (m/z): positive mode 203 [M+H]1+. Purity by HPLC UV (254 nm)-ESI-MS: 99 %. Mp: 210–212 °C.

Di-(5-carboxyl-1H-indole-3-yl)methane (45). The compound was synthesized using indole-5carboxylic acid (16) and was isolated as a brown solid (79 % yield). 1H NMR (600 MHz, DMSO-d6) δ 12.27 (br s, 2H, COOH), 11.12 (br s 2H, NH), 8.17 (d, J = 1.8 Hz, 2H), 7.67 (dd, J = 8.6, 1.7 Hz, 2H), 7.37 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 2.2 Hz, 2H), 4.21 (s, 2H).

13

C NMR (151 MHz, DMSO-d6) δ

168.57, 139.10, 126.81, 124.62, 122.37, 121.58, 120.90, 115.44, 111.24, 39.28. LC-MS (m/z): positive mode 335 [M+H]+-. Purity by HPLC UV (254 nm)-ESI-MS: >98 %. Mp: >300 °C.

Di-(5-benzyloxy-1H-indole-3-yl)methane

(46).

The

compound

was

synthesized

using

5-

benzyloxyindole (17) and was isolated as a brown solid (75 % yield). 1H-NMR (600 MHz; DMSO-d6) δ 10.53 (br s, 2H, NH), 7.39-7.44 (m, 4H), 7.33-7.38 (m, 4H), 7.26-7.32 (m, 2H), 7.20 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 2.4 Hz, 1H, 2-H), 7.07 (d, J = 2.4 Hz, 2H), 6.76 (d, J = 2.5 Hz, 1H), 6.75 (d, J = 2.5 Hz, 1H), 5.02 (s, 4H), 4.02 (s, 2H).

13

C-NMR (151 MHz, DMSO-d6) δ 151.8, 137.9, 131.8, 128.4, 127.8,

127.7, 127.6, 123.6, 114.1, 111.9, 111.5, 102.3, 70.3, 20.9. LC-MS (m/z): positive mode 459 [M+H]+-. Purity by HPLC UV (254 nm)-ESI-MS: >98 %. Mp: 125–127 °C.

Di-(7-methoxy-1H-indole-3-yl)methane (51). The compound was synthesized using 7-methoxyindole (22) and was isolated as a brown solid (93 % yield). 1H NMR (500 MHz, DMSO-d6) δ 10.87 – 10.63 (m, 2H), 7.10 (d, J = 7.9 Hz, 2H), 6.99 (d, J = 2.4 Hz, 2H), 6.84 (t, J = 7.8 Hz, 2H), 6.59 (d, J = 7.6 Hz, 2H), 4.07 (s, 2H), 3.87 (s, 6H). 13C NMR (126 MHz, DMSO-d6) δ 14.20, 20.86, 55.13, 100.55, 101.34, ACS Paragon Plus Environment

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101.49, 111.75, 111.84, 118.68, 122.49, 122.82, 126.50, 128.83, 146.19. LC-MS (m/z): positive mode 307 [M+H]1+. Purity by HPLC UV (254 nm)-ESI-MS: >98 %. Mp: 166-168 °C.

Di-(7-fluoro-1H-indole-3-yl)methane (52). The compound was synthesized using 7-fluoroindole (23) and was isolated as a brown solid (85 % yield). 1H NMR (500 MHz, DMSO-d6) δ 11.21 (br s, 2H, NH), 7.32 (d, J = 1.4 Hz, 2H), 7.20 (d, J = 2.3 Hz, 2H), 6.90 – 6.88 (m, 2H), 6.88 – 6.85 (m, 2H), 4.12 (s, 2H). 13C NMR (126 MHz, DMSO-d6) δ 150.30, 148.37, 131.27, 124.15, 118.49, 114.99, 105.80, 20.92. LC-MS (m/z): positive mode 283 [M+H]1+. Purity by HPLC UV (254 nm)-ESI-MS: 96 %. Mp: 159-161 °C.

Di-(4,5-dichloro-1H-indole-3-yl)methane (53).

The compound was synthesized using 4,6-

dichloroindole (24) and was isolated as a brown solid (83 % yield). 1H-NMR (500 MHz, DMSO-d6) δ 11.21 (br s, 2H, NH), 7.39 (d, J = 2.9 Hz, 2H), 7.03 (d, J = 2.4 Hz, 2H), 6.92 (dd, J = 2.4, 1.1 Hz, 2H), 4.54 (s, 2H).

13

C NMR (126 MHz, DMSO-d6) δ 138.01, 126.41, 125.62, 125.41, 122.47, 119.02,

115.16, 115.63, 110.63, 23.12. LC-MS (m/z): positive mode 385 [M+H]+-. Purity by HPLC UV (254 nm)-ESI-MS: >95 %. Mp: 171–173 °C.

Di-(5-fluoro-6-chloro-1H-indole-3-yl)methane (54). The compound was synthesized using 5-fluoro-6chloroindole (25) and was isolated as a brown solid (76 % yield). 1H NMR (500 MHz, DMSO-d6) δ 10.95 (br s, 2H, NH), 7.46 (d, J = 6.4 Hz, 2H), 7.43 (d, J = 10.3 Hz, 2H), 7.33 (d, J = 2.4 Hz, 2H), 4.05 (s, 2H).

13

C NMR (126 MHz, DMSO-d6) δ 152.32, 150.46, 132.88, 126.16, 126.00, 114.50, 113.20,

112.94, 112.44, 104.96, 104.78, 20.54: LC-MS (m/z): positive mode 349 [M-2H]2-. Purity by HPLC UV (254 nm)-ESI-MS: >98 %. Mp: 225–227 °C.

Di-(4,5-difluoro-1H-indole-3-yl)methane (55).

The

compound

was

synthesized

using

4,5-

difluoroindole (26) and was isolated as a brown oil (87 % yield). 1H NMR (600 MHz, DMSO-d6) δ ACS Paragon Plus Environment


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11.16 (br s, 2H, NH), 7.43 (dd, J = 11.5, 8.0 Hz, 2H), 7.31–7.28 (m, 2H), 7.28–7.24 (m, 2H), 4.04 (s, 2H).

13

C NMR (151 MHz, DMSO-d6) δ 145.80, 144.02, 131.44, 124.92, 124.95, 122.56, 114.38,

105.26, 24.22. LC-MS (m/z): negative mode 317 [M-1H]1-. Purity by HPLC UV (254 nm)-ESI-MS: >95 %. Mp: 117-119 °C.


The

compound

was

synthesized

using

5,6-

difluoroindole (27) and was isolated as a brown solid (83 % yield). 1H NMR (600 MHz, DMSO-d6) δ 7.43 (dd, J = 11.5, 8.0 Hz, 2H), 7.32 – 7.28 (m, 2H), 7.28 – 7.24 (m, 2H), 4.04 (d, J = 1.0 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 147.38, 145.81, 131.45, 124.98, 124.96, 122.57, 105.26, 105.14, 99.34, 99.20, 21.15, 20.67. LC-MS (m/z): negative mode 317 [M-1H]1. Purity by HPLC UV (254 nm)-ESIMS: >98 %. Mp: 122–124 °C.


The

compound

was

synthesized

using

5,7-

difluoroindole (28) and was isolated as a brown solid (80 % yield). 1H NMR (600 MHz, DMSO-d6) δ 11.66 – 11.12 (m, 2H), 7.36 (d, J = 2.5 Hz, 2H), 7.14 (dd, J = 9.5, 2.2 Hz, 2H), 6.88 (dd, J = 11.6, 9.6 Hz, 2H), 4.06 (s, 2H).

13

C NMR (151 MHz, DMSO-d6) δ 156.00, 154.70, 149.20, 147.47, 129.85,

126.16, 121.00, 115.41, 99.68, 95.91, 20.61. LC-MS (m/z): positive mode 331.3 [M+H]1+. Purity by HPLC UV (254 nm)-ESI-MS: 96 %. Mp: 158-160 °C.

Di-(4-fluoro-1H-indole-3-yl)ethane (76). The compound was synthesized by reaction of 4-fluoroindole (6) with acetaldehyde (61) and was isolated as a brown solid (83 % yield). 1H NMR (600 MHz, DMSOd6) δ 10.98 (s, 2H), 7.14 (d, J = 8.1 Hz, 2H), 6.98 (dd, J = 7.9, 5.0 Hz, 2H), 6.90 (d, J = 2.3 Hz, 2H), 6.63 (dd, J = 11.5, 7.8 Hz, 2H), 4.82 (q, J = 7.1 Hz, 1H), 1.66 (d, J = 7.1 Hz, 3H).13C NMR (151 MHz, DMSO-d6) δ 157.33, 155.71, 139.52, 124.74, 122.26, 121.40, 119.56, 114.85, 108.04, 103.46, 29.19, 23.37. LC-MS (m/z): positive mode 295.5 [M+H]1+. Purity by HPLC UV (254 nm)-ESI-MS: >95 %. Mp: 155-157 °C. ACS Paragon Plus Environment

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Di-(indole-3-yl)propane (77). The compound was synthesized by reaction of indole (2) with propionaldehyde and was isolated as a light blue solid (80 % yield); 1H NMR (600 MHz, CDCl3-d) δ 7.89 (s, 2H), 7.57 (d, J = 7.9 Hz, 2H), 7.32 (d, J = 8.2 Hz, 2H), 7.24 (d, J = 6.7 Hz, 2H), 7.12 (t, J = 7.7 Hz, 2H), 7.03 – 6.97 (m, 2H), 4.36 (t, J = 7.5 Hz, 1H), 2.29 – 2.16 (m, 2H), 0.99 (td, J = 7.4, 2.8 Hz, 3H).

13

C NMR (151 MHz, CDCl3-d) δ 13.07, 28.66, 35.89, 76.79, 110.99, 118.96, 119.70, 120.35,

121.40, 121.70, 127.22, 136.58. LC-MS (m/z): negative mode 273 [M-H]-. Purity by HPLC UV (254 nm)-ESI-MS: 96 %. Mp: 122-124 °C.

3,3′-Diindolyl-(p-hydroxyphenyl)methane (83). The compound was synthesized by reaction of indole (2, 10 mmol) with 4-hydroxybenzaldehyde (69, 5 mmol) and was isolated as a red solid (93 % yield). 1H NMR (500 MHz, CDCl3-d) δ 7.91 (s, 1H, NH), 7.38 – 7.31 (m, 2H), 7.29 – 7.24 (m, 2H), 7.16 (dd, J = 8.1, 7.1 Hz, 2H), 7.00 (dd, J = 8.0, 7.0 Hz, 2H), 6.97 – 6.91 (m, 4H), 6.61 (dd, J = 2.4, 1.0 Hz, 2H), 5.86 (s, 1H). 13C NMR (126 MHz, chloroform-d3) δ 160.41, 139.68, 139.65, 136.68, 130.07, 130.01, 126.90, 123.54, 121.99, 119.82, 119.52, 119.27, 115.00, 114.84, 111.07, 39.44. LC-MS (m/z): positive mode 337 [M-2H]2- and negative mode 336 [M-1H]1-. Purity by HPLC UV (254 nm)-ESI-MS: >97 %. Mp: 233–235 °C.

3,3′-Diindolyl-(p-fluorophenyl)methane (85). The compound was synthesized by reaction of indole (2, 10 mmol) with 4-fluorobenzaldehyde (71, 5 mmol) and was isolated as a red solid (80 % yield). 1H NMR (600 MHz, DMSO-d6) δ 10.73 (br s, 2H, NH), 7.32 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 7.9 Hz, 2H), 7.17 – 7.06 (m, 2H), 7.01 (dd, J = 8.2, 6.9 Hz, 2H), 6.84 (dd, J = 8.0, 6.9, Hz, 2H), 6.80 – 6.73 (m, 2H), 6.70 – 6.54 (m, 2H), 5.70 (s, 1H).

13

C NMR (151 MHz, DMSO-d6) δ 155.42, 136.75, 135.36, 129.28,

126.83, 123.53, 120.92, 119.33, 118.84, 118.21, 114.91, 111.53, 59.91. LC-MS (m/z): negative mode 339 [M-1H]1-. Purity by HPLC UV (254 nm)-ESI-MS: >95 %. Mp: 78–80 °C.



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3,3′-Diindolyl-(1,3-benzodioxol-4-yl))methane (86). The compound was synthesized by reaction of indole (2, 10 mmol) with 1,3-benzodioxole-4-carbaldehyde (72, 5 mmol) and was isolated as a yellow solid (91 % yield). 1H NMR (600 MHz, DMSO-d6) δ 10.78 (br s, 2H, NH), 7.33 (dd, J = 8.2, 0.9 Hz, 2H), 7.28 (dd, J = 7.9, 1.1 Hz, 2H), 7.02 (dd, J = 8.0, 6.9 Hz, 2H), 6.89 – 6.84 (m, 2H), 6.83 (m, 3H), 6.78 (d, J = 7.9 Hz, 1H), 5.93 (s, 2H), 5.75 (s, 1H). 13C NMR (151 MHz, DMSO-d6) δ 147.11, 145.31, 139.24, 136.73, 126.73, 123.62, 121.22, 121.00, 119.25, 118.32, 118.31, 111.58, 108.92, 107.90, 107.90, 100.78, 59.90. LC-MS (m/z): positive mode 337 [M-2H]2- and negative mode 336 [M-1H]1-. Purity by HPLC UV (254 nm)-ESI-MS: 99 %. Mp: 106–108 °C.

3-(1H-Indol-3-ylmethyl)-1-methyl-1H-indole (93). The compound was synthesized by reaction of indole (2, 1 mmol) with 1-methylindole (29, 1 mmol) and was isolated as a yellow solid (40 % yield). 1

H NMR (500 MHz, DMSO-d6) δ 10.71 (br s, 1H, NH), 7.52 (dd, J = 18.0, 7.9 Hz, 2H), 7.32 (dd, J =

11.6, 8.2 Hz, 2H), 7.13 (d, J = 2.3 Hz, 1H), 7.09 (dd, J = 8.2, 6.8 Hz, 1H), 7.06 (s, 1H), 7.05 – 6.97 (m, 1H), 6.93 (dd, J = 20.4, 7.4 Hz, 2H), 4.11 (s, 2H), 3.69 (s, 3H).

13

Capt NMR (126 MHz, DMSO-d6) δ

136.89, 136.51, 127.60, 127.25, 122.90, 120.99, 120.86, 118.95, 118.69, 118.22, 118.16, 114.12, 113.76, 111.40, 32.29, 20.79: LC-MS (m/z): positive mode 261 [M+H]1+. Purity by HPLC UV (254 nm)-ESI-MS: 97 %. Mp:125–127 °C.

3-(1H-Indol-3-ylmethyl)-5-methoxy-1H-indole (94). The compound was synthesized by reaction of indole (2, 1 mmol) with 5-methoxylindole (8, 1 mmol) and was isolated as a brown solid (47 % yield). 1

H NMR (500 MHz, DMSO-d6) δ 10.70 (br s, 1H, NH), 7.64 (d, J = 7.5 Hz, 1H), 7.34 (d, J = 8.1 Hz,

1H), 7.23 (d, J = 9.2 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.07 (s, 1H), 6.89 (s, 2H), ), 6.86 (d, J = 8.6 Hz, 1H), 4.21 (s, 2H) 3.81 (s, 3H).

13

C NMR (151 MHz, DMSO-d6) δ 153.91,

136.62, 131.70, 128.04, 127.73, 123.27, 122.39, 122.01, 119.34, 119.25, 115.72, 115.41, 112.24,


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111.92, 111.21, 101.23, 56.03, 21.41. LC-MS (m/z): positive mode 297 [M+H]1+. Purity by HPLC UV (254 nm)-ESI-MS: 95 %. Mp: 102–104 °C.

3-(1H-Indol-3-ylmethyl)-5-fluoro-1H-indole (95). The compound was synthesized by reaction of indole (2, 1 mmol) with 5-fluoroindole (9, 1 mmol) and was isolated as a brown solid (41 % yield). 1H NMR (500 MHz, DMSO-d6) δ 10.89 – 10.46 (m, 2H, NH), 7.50 (d, J = 7.8 Hz, 1H), 7.33 – 7.26 (m, 2H), 7.21 (dd, J = 5.2, 2.6 Hz, 2H), 7.16 (d, J = 2.3 Hz, 1H), 7.02 (dd, J = 8.1, 6.9 Hz, 1H), 6.91 (dd, J = 7.9, 6.9 Hz, 1H), 6.85 (dd, J = 9.2, 2.6 Hz, 1H), 4.09 (s, 2H).

13

Capt NMR (126 MHz, DMSO-d6) δ

155.66, 136.53, 133.17, 127.26, 125.02, 122.92, 122.85, 120.87, 120.83, 118.74, 118.15, 118.12, 114.66, 114.00, 112.32, 112.25, 111.42, 20.94. LC-MS (m/z): positive mode 263 [M-H]1-. Purity by HPLC UV (254 nm)-ESI-MS: 97 %. Mp: 141–143 °C.

Methyl 3-((1H-indol-3-yl)methyl)-1H-indole-5-carboxylate (96). The compound was synthesized by reaction of indole (2, 1 mmol) with methyl indole-5-carboxylate (14, 1 mmol) and was isolated as a brown solid (35 % yield). 1H NMR (600 MHz, DMSO-d6) δ 10.72 (br s, 2H, NH), 8.20 (d, J = 1.5 Hz, 1H), 7.68 (dd, J = 8.5, 1.7 Hz, 1H), 7.50 (dd, J = 8.0, 1.1 Hz, 1H), 7.39 (d, J = 8.5 Hz, 1H), 7.31 (dd, J = 8.3, 0.9 Hz, 1H), 7.26 (d, J = 2.2 Hz, 1H), 7.09 – 7.00 (m, 2H), 6.91 (dd, J = 8.0, 7.0, Hz, 1H), 4.18 (s, 2H), 3.79 (s, 3H, CH3).

13

C NMR (151 MHz, DMSO-d6) δ 167.45, 139.19, 136.57, 127.23, 126.91,

124.88, 122.88, 122.00, 121.45, 120.98, 119.76, 118.74, 115.87, 113.96, 111.49, 111.44, 51.71, 20.90. LC-MS (m/z): positive mode 305 [M+H]1+. Purity by HPLC UV (254 nm)-ESI-MS: 95 %. Mp: 146– 148 °C.

3-((1H-indol-3-yl)methyl)-1H-indole-5-carboxylic acid (97). To a solution of compound 96 (10 mmol) in ethanol (10 mL) was added 2N NaOH (2 mL) and the resulting mixture was heated to reflux for 1 h. After completion of the reaction, ethanol was removed under reduced pressure, the pH was adjusted to 4–5, and the product was filtered, washed with H2O and dried under vacuum. The compound ACS Paragon Plus Environment


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was isolated as a light brown solid (99 % yield). 1H NMR (600 MHz, DMSO-d6) δ 12.25 (s, 1H, COOH), 10.73 (br s, 1H, NH), 8.19 (s, 1H), 7.66 (dd, J = 8.4, 1.6 Hz, 1H), 7.50 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.24 (d, J = 2.3 Hz, 1H), 7.09 (d, J = 2.3 Hz, 1H), 7.02 (t, J = 7.6 Hz, 1H), 6.91 (d, J = 7.5 Hz, 1H), 4.16 (s, 2H).

13

C NMR (151 MHz, DMSO-d6) δ 168.48

(COOH), 139.05, 136.56, 127.24, 126.86, 124.59, 122.88, 121.58, 120.95, 118.75, 118.22, 115.76, 114.00, 111.48, 111.16, 20.96. LC-MS (m/z): positive mode 291 [M+H]1+. Purity by HPLC UV (254 nm)-ESI-MS: 98 %. Mp: 244–246°C.

(E/Z)-3-((5-Fluoro-1H-indol-3-yl)methylene)indoline-2-one (105). The compound was synthesized by reaction of 2-oxindole (98, 37 mmol) with 5-fluoroindole-3-carbaldehyde (101, 41 mmol) and was isolated as a yellow solid (69 % yield). 1H NMR (500 MHz, DMSO-d6) δ 8.10 (s, 1H), 8.03 (dd, J = 10.1, 2.5 Hz, 1H), 7.90 (dd, J = 7.6, 1.1 Hz, 1H), 7.51 (dd, J = 8.8, 4.6 Hz, 1H), 7.13 (dd, J = 7.5, 1.1 Hz, 1H), 7.06 (dd, J = 9.1, 2.5 Hz, 1H), 6.98 (dd, J = 7.5, 1.0 Hz, 1H), 6.86 – 6.78 (m, 2H). 13Capt NMR (126 MHz, DMSO-d6) δ 168.20, 159.33, 139.35, 135.14, 132.60, 129.28, 127.11, 126.98, 125.78, 120.64, 119.51, 119.05, 113.54, 113.47, 111.66, 110.73, 110.52. LC-MS (m/z): positive mode 279 [M+H]1+. Purity by HPLC UV (254 nm)-ESI-MS: 98 %. Mp: 279–281 °C.

(E/Z)-3((5-Methoxy-1H-indol-3-yl)methylene)-5-fluoroindoline-2-one (108). The compound was synthesized by reaction of 5-fluoro-2-oxindole (99, 37 mmol) and 5-methoxyindole-3-carbaldehyde (102, 41 mmol) and was isolated as a yellow solid (80 % yield).1H NMR (500 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.19 (s, 1H), 7.86 (dd, J = 9.4, 2.6 Hz, 1H), 7.76 (d, J = 2.4 Hz, 1H), 7.40 (d, J = 8.7 Hz, 1H), 6.92 (dd, J = 9.7, 8.4 Hz, 1H), 6.86 (dd, J = 8.7, 2.4 Hz, 1H), 6.79 (dd, J = 8.4, 4.5 Hz, 1H), 3.88 (s, 3H).13C NMR (126 MHz, DMSO-d6) δ 168.35, 159.03, 155.32, 135.27, 134.75, 131.07, 130.91, 129.92, 129.40, 129.38, 129.34, 127.65, 118.01, 114.25, 113.53, 113.14, 112.66, 112.61, 112.47, 111.56, 109.92, 109.55, 109.49, 109.25, 106.16, 105.95, 101.06, 55.80: LC-MS (m/z): positive mode 309 [M+H]1+. Purity by HPLC UV (254 nm)-ESI-MS: 96 %. Mp: 296–298°C. ACS Paragon Plus Environment

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(E/Z)-3-((1H-Indol-5-yl)methylene)indoline-2-one (109). The compound was synthesized by reaction of 2-oxindole (98, 37 mmol) with indole-5-carbaldehyde (103, 41 mmol) and was isolated as a brown solid (73 % yield). 1H NMR (500 MHz, DMSO-d6) δ 11.37 (d, J = 19.4 Hz, 1H), 10.50 (s, 1H), 8.85 – 8.80 (m, 0H), 8.30 (dd, J = 8.7, 1.6 Hz, 0H), 7.96 (d, J = 1.5 Hz, 1H), 7.88 (s, 0H), 7.76 (d, J = 7.1 Hz, 2H), 7.69 (d, J = 7.6 Hz, 0H), 7.57 – 7.47 (m, 2H), 7.47 – 7.36 (m, 2H), 7.20 (td, J = 7.7, 1.1 Hz, 1H), 7.15 (td, J = 7.6, 1.1 Hz, 0H), 6.97 (td, J = 7.5, 0.9 Hz, 0H), 6.90 – 6.84 (m, 2H), 6.84 – 6.77 (m, 1H), 6.60 – 6.48 (m, 1H), 5.74 (s, 0H). 13Capt NMR (126 MHz, DMSO-d6) δ 169.26, 167.61, 155.60, 142.62, 140.09, 139.76, 138.80, 137.39, 136.86, 129.45, 127.86, 127.73, 126.99, 126.79, 126.34, 126.19, 125.96, 125.75, 125.20, 124.75, 123.04, 122.89, 122.82, 121.90, 121.70, 121.06, 120.89, 119.04, 111.86, 111.23, 110.04, 109.17, 102.62, 102.17, 46.78. LC-MS (m/z): positive mode 261 [M+H]1+Purity by HPLC UV (254 nm)-ESI-MS: >98 %. Mp: 153–155 °C.

3-((5-Methoxy-1H-indol-3-yl)methyl)indolin-2-one (110). The compound was synthesized from 106 (5 mmol) and was isolated as a light yellow solid (89 % yield). 1H NMR (500 MHz, DMSO-d 6) δ 10.57 – 10.52 (m, 1H), 10.20 (s, 1H), 7.15 (d, J = 8.7 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 7.01 – 6.96 (m, 2H), 6.86 (d, J = 2.5 Hz, 1H), 6.81 (td, J = 7.5, 1.0 Hz, 1H), 6.69 (d, J = 7.7 Hz, 1H), 6.65 (dd, J = 8.7, 2.5 Hz, 1H), 3.76 (dd, J = 7.6, 4.7 Hz, 1H), 3.72 (s, 3H), 3.40 – 3.33 (m, 1H), 3.08 (dd, J = 14.7, 7.5 Hz, 1H).

13

Capt NMR (126 MHz, DMSO-d6) δ 178.78, 152.99, 142.91, 131.18, 129.96, 127.66, 127.49,

124.43, 124.31, 120.94, 111.89, 111.11, 109.03, 100.59, 55.39, 46.00, 25.50. LC-MS (m/z): positive mode 291 [M+H]1+. Purity by HPLC UV (254 nm)-ESI-MS: 96 %. Mp: 223–225 °C.

5-Fluoro-3-((5-fluoro-1H-indol-3-yl)methyl)indolin-2-one (111). The compound was synthesized using 107 (5 mmol) and was isolated as a yellow solid (96 % yield). 1H NMR (500 MHz, DMSO-d6) δ 11.13 – 10.52 (m, 1H), 10.21 (s, 1H), 7.26 (dd, J = 13.1, 3.6 Hz, 2H), 6.99 (d, J = 2.5 Hz, 1H), 6.95 (dd, J = 8.6, 2.8 Hz, 1H), 6.86 (dd, J = 18.5, 9.1, 2.7 Hz, 2H), 6.63 (dd, J = 8.4, 4.4 Hz, 1H), 3.79 (t, J = 5.8 ACS Paragon Plus Environment


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Hz, 1H), 3.37 (dd, J = 14.6, 5.0 Hz, 1H), 3.16 (dd, J = 14.6, 6.6 Hz, 1H). 13C NMR (126 MHz, DMSOd6) δ 178.75, 158.69, 157.70, 156.82, 155.87, 139.16, 132.63, 125.95, 113.78, 113.60, 112.38, 112.27, 112.19, 109.60, 109.53, 109.18, 108.98, 103.56, 103.37, 46.72, 24.91. LC-MS (m/z): positive mode 299 [M+H]1+. Purity by HPLC UV (254 nm)-ESI-MS: 98 %. Mp: 154–156 °C.

5-Fluoro-3-((5-methoxy-1H-indol-3-yl)methyl)indolin-2-one (112). The compound was synthesized using 108 (5 mmol) and was isolated as a brown solid (91 % yield). 1H NMR (500 MHz, DMSO-d6) δ 10.56 (d, J = 2.2 Hz, 1H), 10.20 (s, 1H), 7.15 (d, J = 8.8 Hz, 1H), 7.00 (d, J = 2.4 Hz, 1H), 6.93 – 6.81 (m, 3H), 6.67 – 6.58 (m, 2H), 3.83 – 3.74 (m, 1H), 3.71 (s, 3H), 3.41 – 3.32 (m, 1H), 3.12 (dd, J = 14.6, 7.1 Hz, 1H).

13

Capt NMR (126 MHz, DMSO-d6) δ 178.75, 158.62, 156.75, 153.06, 139.17, 131.17,

127.64, 124.50, 113.73, 113.55, 112.45, 112.26, 111.96, 111.25, 110.04, 109.56, 109.49, 100.62, 55.38, 46.70, 25.21: LC-MS (m/z): positive mode 311 [M+H]1+. Purity by HPLC UV (254 nm)-ESI-MS: >98 %. Mp: 162–164 °C.

3-((1H-Indol-5-yl)methyl)indolin-2-one (113). The compound was synthesized using 109 (5 mmol) and was isolated as a brown (95 % yield). 1H NMR (500 MHz, DMSO-d6) δ 10.91 (s, 1H), 10.22 (s, 1H), 7.31 – 7.22 (m, 2H), 7.20 (d, J = 8.2 Hz, 1H), 7.05 (dd, J = 7.2, 2.4 Hz, 1H), 6.88 (dd, J = 8.2, 1.8 Hz, 1H), 6.78 (d, J = 6.6 Hz, 2H), 6.68 (d, J = 7.7 Hz, 1H), 6.28 (t, J = 2.5 Hz, 1H), 3.76 (dd, J = 8.0, 4.9 Hz, 1H), 3.38 (dd, J = 13.9, 5.0 Hz, 1H), 2.96 (dd, J = 13.8, 8.0 Hz, 1H).

13

Capt NMR (126 MHz,

DMSO-d6) δ 178.44, 142.77, 134.81, 129.46, 128.23, 127.68, 127.50, 125.37, 124.51, 122.71, 120.90, 120.53, 110.98, 109.13, 100.88, 47.25, 35.75. LC-MS (m/z): positive mode 263 [M+H]1+. Purity by HPLC UV (254 nm)-ESI-MS: 99 %. Mp: 182–184 °C.

Biological assays The recombinant CHO cell line expressing the human GPR84 (CHO-hGPR84 cells) with a βgalactosidase fragment and arrestin containing the complementary fragment of the enzyme for ACS Paragon Plus Environment

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performing β-arrestin recruitment assays based on enzyme complementation technology with a luminiscent readout (Pathhunter®) was purchased from DiscoverX (Fremont, CA). This cell line was used for the β-arrestin recruitment assays as well as for cAMP accumulation assays. The CHO-hGPR84 cells were cultured in F12 medium supplemented with 10 % FCS, 100 units/mL penicillin G, 100 µg/mL streptomycin, 800 µg/mL G 418, 300 µg/mL hygromycin B, and 1 % ultraglutamin (Invitrogen, Carlsbad, CA or Sigma-Aldrich, St. Louis, MO). Stock solutions of compounds including forskolin were prepared in DMSO. The final DMSO concentration in the assays did not exceed 1%. Data analysis was performed with GraphPad Prism (Version 6.02). Concentration-response data were fitted by nonlinear regression to estimate EC50 values (Prism 6.02). Statistical analysis was performed using GraphPad Prism 6.02. Differences between means were calculated using two-tailed Student’s t test and a p-value of

< 0.05 was considered statistically significant. The cooperativity factor α and the

equilibrium dissociation constant Kb for allosteric modulation were calculated by fitting the data to the “allosteric EC50 shift” equation (Prism 6.02).

GPR84 cAMP accumulation assays CHO-hGPR84 cells were cultured on 24-well plates for 24 h. The culture medium was removed and cells were incubated with Hanks Balanced Salt Solution (HBSS) buffer (pH 7.4) at 37°C in presence of the cAMP phosphodiesterase inhibitor RO-20-1724 (final concentration of 40 µM). Cells were then stimulated by addition of forskolin (10 µM) in the absence (control) or presence of test compounds for 15 min. After removal of the buffer, cells were lyzed by the addition of a hot (90 °C) lysis solution containing 4 mM EDTA, and 0.01 % Triton X-100 in water. AMP levels in the supernatant were quantified by a radiometric assay using a cAMP binding protein prepared from bovine adrenal medulla and [³H]cAMP (Perkin-Elmer, Rodgau, Germany) as described previously.90 The forskolin-induced increase in cAMP concentration in the presence of agonists was expressed as percentage of the response to forskolin in the absence of agonists (% of control). Three independent experiments were performed, each in duplicates. ACS Paragon Plus Environment


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GPR84 β-arrestin recruitment assays β-Arrestin assays were performed as previously described.91 CHO-hGPR84 cells (20,000 per well) in 90 µL of Cell Plating 2 Reagent (DiscoverX®) were seeded into 96-well plates (Thermo Scientific, Waltham, MA). Compound dilutions (in DMSO, 10 µL per well) were added to the cells. The final DMSO concentration did not exceed 1 %. After 90 min of incubation, 50 µL of detection reagent (DiscoverX®) per well were added. After 60 min of incubation at rt the luminescence was measured using an NXT plate reader (Perkin-Elmer, Rodgau, Germany). Three to five independent experiments were performed, each in duplicates.

FFAR4 β-arrestin recruitment assays A parental CHO β-arrestin cell line and the corresponding expression vectors were purchased from DiscoverX (Freemont, CA). After subcloning of the human FFAR4 (GPR120) cDNA sequence into a suitable expression vector, the parental cell line was transfected with the DNA construct by lipofection. Using these cells recombinantly expressing the human FFAR4 the assay was essentially performed as previously described.91 In brief, on the day before the assay the cells were seeded into a white 96-well plate at a density of 30,000 cells per well. Approximately four hours prior to the assay the medium was exchanged for 90 µl of serum-free F12 medium per well. Test compounds were diluted in DMSO followed by a subsequent dilution step in serum-free medium (resulting in a final concentration of 1% DMSO in the assay). In antagonist assays test compound dilutions (5 µl per well) were added 30 min before the addition of the reference agonist 4-[(4-fluoro-4'-methyl[1,1'-biphenyl]-2-yl)methoxy]benzenepropanoic acid (TUG-891, 5 µl per well, final concentration 4 µM ≈ EC80). In agonist assays 10 µl of test compound dilutions were added. After 90 min in the presence of an agonist, 50 µl of a detection reagent was added to each well. After an incubation of 60 min at rt in the dark, luminescence in each well was measured using an NXT plate reader (Perkin-Elmer, Meriden, CT). All compounds were tested at a final concentration of 10 µM. Test results were normalized to values obtained by ACS Paragon Plus Environment

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determining the background and the signal induced by 30 µM and 4 µM of TUG-891 in agonist and antagonist assays, respectively. Three to four independent experiments were performed in duplicates.

FFAR1 calcium mobilization assay To generate a cell line for calcium mobilization assays the cDNA sequence of FFAR1 (GPR40) was inserted into the retroviral plasmid pLXSN. The retroviral transfection of 1321N1 astrocytoma cells was performed as previously described.90 On the day before the assay the cells recombinantly expressing FFAR1 were seeded into 96-well plates (black, clear bottom) at a density of 50,000 cells per well. The cells were cultured in DMEM medium supplemented with 10 % FCS, 100 units/mL penicillin G, 100 µg/mL streptomycin and 800 µg/mL G418. On the day of the assay the medium was exchanged for 40 µl of a HBSS buffer solution containing 3 µM of the calcium dye Fluo-4-AM (Life Technology, Darmstadt, Germany) and 0.06 % Pluronic F-127. After 60 min of incubation at rt in the dark the dye solution was exchanged for 190 µl and 189 µl of HBSS buffer, in agonist and antagonist assays, respectively. Using a FlexStation® 3 plate reader (Molecular Devices, Sunnyvale, CA) 10 µl of test compound solution were added to each well. In antagonist assays cells were preincubated for 30 min with

test

compound

solutions

(1

µl

per

well)

before

the

reference

agonist

3-(4-(o-

tolylethynyl)phenyl)propanoic acid (TUG-424) was added (10 µl per well, final concentration: 1 µM ≈ EC80). The final DMSO concentration did not exceed 1 %. Fluorescence was measured at 520 nm (excitation 485 nm) for 90 intervals of 1.2 s each. All compounds were tested at a final concentration of 10 µM. Signals induced by the test compounds were normalized to the signals induced by 1 µM and 10 µM of TUG-424, in antagonist and agonist assays, respectively. Three to four independent experiments were performed in duplicates.

Arylhydrocarbon receptor (AhR) activation



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HepG2 cells (3x105 cells/ml) were stimulated for 18 h with 5 µM or 20 µM of diindolylmethane, 38, 57, or left untreated. 3-Methylcholanthrene (3MC, 10 µM) was used as a positive control. RNA was extracted using the Quick RNATM Mini prep kit (Zymo Research, Irvine, CA, U.S.A.) according to the manufacturer's instructions. First-strand cDNA was synthesized from 500 ng of total RNA using oligo(dT)12–18 primers and RevertAid reverse transcriptase (Thermo Fischer Scientific, Schwerte, Germany ). Real time PCR was performed in a CFX96 Real time System (Biorad, Hercules, CA, USA) using absolute SYBR-green ROX master mix (Thermo Fisher Scientific). Cyp1A1 expression levels were normalized to gapdh and were displayed as fold-change relative to untreated samples used as the calibrator (set to 1). Primers for cyp1A1 and gapdh were: cyp1A1-fwd: 5´-cag gta tgt ggt ggt atc agg-3´ and cyp1A1-rev: 5´-ggt agg tag cga aga ata gg-3´; gapdh-fwd: agc cac atc gct cag aca c and gaphd-rev: gcc caa tac gac caa atc c-3´. Three independent experiments were performed.

Determination of stability Stability of selected compounds in acidic, neutral and basic solutions was measured using LC-MS analysis.89 The artificial gastric acid was prepared as previously described.92,93 Pepsin (3.2 g), NaCl (2.0 g) and HCl (1 M, 80 mL) were mixed with pure water to obtain 1000 mL (pH 1.2), and the resulting solution was stored at 4 °C. Stock solutions (2.5 mM) of 1, 38, and 52 were prepared in a mixture of methanol/acetonitrile (1:1) and 40 µL of stock solution was added to 960 µL of water or 0.01% aqueous ammonia solution (pH 9), or 1 mM sodium hydroxide solution (pH 11) or artificial gastric acid yielding a final compound concentration of 100 µM. The solutions were incubated at 37 oC for up to 24 h and subsequently analyzed at regular time intervals. Mass spectra were recorded on a micrOTOF-Q mass spectrometer (Bruker) with an ESI-source coupled with an HPLC Dionex Ultimate 3000 (Thermo Scientific) using an EC50/2 Nucleodur C18 Gravity 3 µm column (Macherey-Nagel). The column temperature was 25 °C. The HPLC separation started with 90 % water containing 2 mM ammonium acetate and 10 % acetonitrile. A gradient started afeter 1 min to 100 % acetonitrile within 9 min. The


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column was flushed with 100 % acetonitrile for an additional period of 5 min. Sample solution (5 µL) was injected at a flow rate of 0.3 mL/min. Positive full scan MS was observed from 50-1000 m/z.

ASSOCIATED CONTENT Supporting Information Melting points and purities of GPR84 agonists; potency of GPR84 agonists at the human fatty acid receptors FFAR1 and FFAR4 and the human GPR35; evaluation of selected arylhydrocarbon receptor ligands at GPR84 in cAMP assays; analysis of compound stability; 1H,

13

C /

13

Capt-NMR spectra of

compounds 1, 34, 35, 38, 51-57, 93 and 95; molecular string formulae.

AUTHOR INFORMATION Corresponding Author *Dr. Christa E. Müller Pharmazeutisches Institut Pharmazeutische Chemie I An der Immenburg 4, D-53121 Bonn, Germany Phone:

+49-228-73-2301

Fax:

+49-228-73-2567

E-mail: [email protected]

ACKNOWLEDGMENTS T.P. is grateful to the Alexander von Humboldt (AvH) foundation and to Bayer Pharma for supporting a postdoctoral fellowship. G.B. thanks the CAPES Foundation and the Ministry of Education of Brazil for supporting an internship. I.F. is a member of the DFG-funded Cluster of Excellence ImmunoSensation. ACS Paragon Plus Environment


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We thank Marion Schneider for LCMS analyses, Sabine Terhart-Krabbe and Annette Reiner for NMR studies and Daniel Müller for performing some of the β-arrestin assays.

ABBREVIATIONS USED GPCR, G protein-coupled receptor; cAMP, cyclic adenosine monophosphate; GPR, G protein-coupled receptor; FFA/R, free fatty acid/receptor; M/L/SCFA, medium/long/short chain fatty acid; GLP-1, glucagon like peptide-1; PTX, pertussis toxin; Th, T helper; TB, tuberculosis; TNF, tumor necrosis factor; TM, transmembrane; CHO, Chinese hamster ovary; SAR, structure-activity relationship; DIM, Diindolylmethane; SDS, sodium dodecylsulfate; CTAB, N-acetyl-N,N,N-trimethylammonium bromide; MW, microwave; DCE, dichloroethane; DMSO, dimethyl sulfoxide; TFAA, trifluoroacetic acid; NMR, nuclear magnetic resonance; HPLC, high performance liquid chromatography; ESI, electrospray ionization; EA, ethyl acetate; DCM, dichloromethane; TLC, thin layer chromatography; PE, petrolether; DMF, N,N’-dimethylformamide; THF, tetrahydrofuran.

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